DOI:http://dx.doi.org/10.7314/APJCP.2014.15.4.1711 Effects of Withaferin A on A549 Cellular Proliferation and in Non-small Cell Lung Cells

RESEARCH ARTICLE

Effect of Withaferin A on A549 Cellular Proliferation and Apoptosis in Non-small Cell Yong Cai1, Zhao-Ying Sheng1, Yun Chen1, Chong Bai2*

Abstract

Objective: To explore the effect of Withaferin A on A549 cellular proliferation and apoptosis in non-small cell lung cancer (NSCLC). Materials and Methods: NSCLC cell line A549 was selected to explore the effect of Withaferin A on A549 cellular proliferation, apoptosis and the PI3K/Akt signal pathway capable of regulating tumor biological behavior by assessment of cellular proliferation, cellular apoptotic rates and cellular cycling as well as by immuno-blotting. Results: Withaferin A could inhibit A549 cellular proliferation and the control rate was dosage-dependent (P<0.05), which also increased time-dependently with the same dosage of Withaferin A (P<0.05). The apoptotic indexes in A549 cells treated with 0, 2.5, 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A for 48 h were significantly different (P<0.05). In addition, the apoptotic rates of each group in both early and advanced stages were higher than those in 0 μmol·L-1 (P<0.05), which were evidently higher after 48 h than those after 24 h (P<0.05). A549 cells treated by Withaferin A for 48 h were markedly lower in Bcl-2 level and obviously higher in Bax and cleaved -3 levels than those treated by 0 μmol·L-1 Withaferin A (P<0.05), and there were significant differences among 5, 10 and 20 μmol·L-1 Withaferin A (P<0.05). The ratios of A549 cells -1 treated by Withaferin A for 48 h in G0/G1 stage were higher than those in 0 μmol·L , while those in S and G2/M stages were obviously lower than those in G2/M stage, and there were significant differences in 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A (P<0.05). Additionally, p-Akt/Akt values were in reverse association with dosage, and the differences were significant (P<0.05). Conclusion: Withaferin A can inhibit the proliferation and apoptosis of A549 cells by suppressing activation of the PI3K/Akt pathways.

Keywords: Non-small cell lung cancer - proliferation - apoptosis - A549 cells

Asian Pac J Cancer Prev, 15 (4), 1711-1714

Introduction proliferation, apoptosis and PI3K/Akt signal pathways capable of regulating tumor biological behaviors, and the Lung cancer is the leading cause for tumor-associated results are as follows. deaths, in which non-small cell lung cancer (NSCLC) accounts for 85% and most patients are in advanced stages Materials and Methods with poor therapeutic efficacy when diagnosed (Jemal et al., 2010; Cho et al.,2013; Lu et al., 2013; Wang et Agents al., 2013; Yan et al., 2013; Kim et al., 2014). Though A549 cell lines were obtained from American ATCC; there has been great development in clinical treatment of Withaferin A, dimethyl sulfoxide (DMSO), methyl NSCLC, its prognosis is still unsatisfactory due to the low thiazolyl tetrazolium (MTT), Sodium dodecyl sulfate rate of 5-year survival in clinic (Verdecchia et al., 2007). (SDS) and Tris were purchased from American Sigma- Therefore, seeking more effective drugs for NSCLC is of Aldrich company; RPMI 1640 culture medium, Fetal calf great significance in its clinical treatment. Withaferin A is Serum (FCS) and trypsase were brought from American an active ingredient extracted from Withania omnifera (Yu Gibco company; penicillin and Streptomycin were gained et al., 2013), which possesses the functions of regulating from Hyclone company; glycine, RNase, Acrylamide and immunity and anti-, etc.. In recent years, it methylene-bisacrylamide were received from American has also been discovered that Withaferin A has anti-tumor Amresco company; Annexin-FITC/PI kits for apoptotic activity and is effective in inhibiting multiple tumors, detection was brought from Keygen Biotech. Co., Ltd; such as , etc. (Hahm et al., 2013; Lee et al., internal references GAPDH, Bcl-2 and Cleaved caspase-3 2013). NSCLC cell line A549 was selected in this study polyclonal antibodies were obtained from American to explore the effect of Withaferin A on A549 cellular Cell Sigaling Technology company; Bax, Akt and P-Akt

1Department of Radiation Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, 2Department of Respiration, Changhai Hospital, Second Military Medical University, Shanghai, China *For correspondence: [email protected]

Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 1711 Yong Cai et al Table 1. Effect of Withaferin A on Cellular Apoptosis (%, χ±s) Groups 24 h 48 h Early stage Advanced stage Early stage Advanced stage 0 μmol·L-1 3.18±0.53 1.16±0.73 4.42±1.26 2.21±0.93 2.5 μmol·L-1 6.72±1.90a 3.25±1.19a 11.63±2.48ab 6.38±1.42ab 5.0 μmol·L-1 11.91±2.91a 6.84±1.35a 13.57±2.35a 9.27±1.55ab 10.0 μmol·L-1 17.08±4.18a 8.29±2.51a 22.29±4.70ab 10.65±2.82a 20.0 μmol·L-1 21.37±3.37a 11.68±2.94a 28.68±5.58ab 15.63±2.73ab Compared with 0 μmol·L-1, aP<0.05; Compared with 24 h, bP<0.05 Figure 1. Control Rate of Cellular Proliferation A for 48 h were collected and centrifuged at high-speed, Treated with Different Dosages of Withaferin A supernatant was discarded and cells were decomposed polyclonal antibodies were purchased from Santa Cruz on ice to determine the protein levels by BAC method. company; and Hoechst 33258 was brought from Beyotime The protein samples were mixed with loading buffer and Institute of Biotechnology. given 10% SDS-PAGE gel electrophoresis under routine methods. The constant voltages of spacer and separation Cellular proliferation detection gels were 80 V (for 30 min) and 100 V (about 80 min), A549 cells in logarithmic phase were treated by 0.25% respectively. Then, the samples were transformed to trypsase, made into mono-cellular suspension by blowing PVDF membrane in semi-dry condition, added with and beating, inoculated in 96-well plate by 1×105/well appropriate amounts of p-Akt primary antibodies (1: for totally 200 μL and cultured for 24 h, added with 20 200), Bax, Caspase-3 and Bcl-2 (1: 300), incubated at μL 0, 2.5, 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A and 4℃ overnight, added with secondary antibodies (1: 3000) cultured for 24, 48, 72 and 96 h respectively in incubators and incubated for 2 h in room temperature by shaking, containing 5% CO2 saturated humidity at 37℃, added with which were colored by ECL and developed to analyze 10 μL 5 mg·mL-1 MTT solution in each well, and cultured the optical intensity of each developed stripe by Gel-Pro for 4 h. Optical density A492 in each well under 492 nm analyzer. p-Akt was the ratio of solved solution with Akt was measured by ELIASA and it was set up as 100% in while the relevant expressions of other proteins were the control group. Three parallel holes were established in ratios with GAPDH. each group and the study was repeated for 3 times to obtain the mean values. Control rate (%)=[ (control group-blank Statistical data analysis group)- (medicine group-blank group)]/ ( control group- Windows SPSS 16.0 software was applied to analyzed blank group)×100%. data expressed by Mean±Standard Deviation (χ±s), while one way ANOVA was used for multi-group comparisons Apoptotic index detection and SNK method for paried-comparison. P<0.05 was A549 mono-cellular suspension was inoculated on regarded as statistically significant. sterilized slide placed in the bottom of 12-well culture plate, and culture medium was discarded after cellular Results adherence presented. Then culture mediums containing different dosages of Withaferin A were added and Effect of Withaferin A on cellular proliferation cultured for 48 h, added with paraformaldehyde to fix A549 cells were treated by 0, 2.5, 5.0, 10.0 and 20.0 cells, washed by PBS, added with 5 μg·mL-1 Hoechst μmol·L-1 Withaferin A for 24 h, 48 h, 72 h and 96 h, 33258 and stained for 30 min at room temperature respectively, and the MTT laboratory results showed that shielded from light. Fluorescent microscope was used Withaferin A could inhibit the proliferation of A549 cells, to observe cellular morphology and 5 high-power fields which was in positive association with the dosages and in were selected to calculate the counts of apoptotic cells and dosage-dependent relationship (P<0.05). In addition, in total cells, so as to obtain the apoptotic indexes. Apoptotic the same dosage of Withaferin A, the cellular control rate index=Apoptotic cell count/Total cell count×100%. also increased time-dependently (P<0.05) (Figure 1).

Detections of cellular apoptotic rate and cellular cycle Effect of Withaferin A on cellular apoptosis A549 cells were inoculated in 6-well culture plate Cellular apoptotic indexes: The apoptotic indexes of by 1×106/well, added with 20 μL 0, 2.5, 5.0, 10.0 and A549 cells treated respectively by 0, 2.5, 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A and cultured for 24 h in 5% 20.0 μmol·L-1 Withaferin A for 48 h were (2.75±0.64)%,

CO2 incubator at 37℃ respectively, washed by PBS (4.61±1.36)%, (9.75±2.78)%, (12.92±3.42)% and once based on the instruments of apoptosis detection kit, (18.68±4.31)% respectively, and the differences were all added with Annexin V/FITC and PI, and stained for 15 significant P( <0.05) (Figure 2). min to calculate apoptotic rate and cellular cycle by flow Cellular apoptotic rate: A549 cells were treated by cytometry. 0, 2.5, 5.0, 10.0 and 20.0μmol·L-1 Withaferin A for 24 h and 48 h respectively, and the Annexin-FITC/PI double- Immuno-blotting method staining method indicated that the apoptotic rates in early A549 cells treated by different dosages of Withaferin and advanced stages of each dosage were all evidently 1712 Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 DOI:http://dx.doi.org/10.7314/APJCP.2014.15.4.1711 Effects of Withaferin A on A549 Cellular Proliferation and Apoptosis in Non-small Cell Lung Cancer Cells

Figure 2. Primary Figures of Apoptosis after Treatment in each Group

Table 2. Effect of Withaferin A on Apoptosis- Table 3. Cellular Cycle Distribution after 48 h100.0 Associated Gene Expression (%, χ±s) Treatment with Withaferin A (%, χ±s) 6.3 12.8 10.1 20.3 Groups Bcl-2 Bax Cleaved caspase-3 10.3 Groups G0/G1 S G2/M 0 μmol·L-1 0.72±0.23 0.32±0.09 0.28±0.14 0 μmol·L-1 42.76±4.28 39.87±3.29 16.40±2.38 75.0 25.0 30.0 2.5 μmol·L-1 0.68±0.19 0.36±0.18 0.35±0.19 2.5 μmol·L-1 47.76±2.59 33.52±3.52 12.40±2.25 -1 a a a -1 a a a 75.0 5.0 μmol·L 0.46±0.17 0.43±0.20 0.47±0.22 5.0 μmol·L 52.90±3.32 29.16±1.79 8.15±1.26 56.3 46.8 -1 a a a -1 a a a 51.1 10.0 μmol·L 0.33±0.13 0.52±0.21 0.54±0.27 10.0 μmol·L 61.17±4.79 27.58±1.84 7.28±2.68 51.7 20.0 μmol·L-1 0.27±0.09a 0.60±0.24a 0.67±0.20a 20.0 μmol·L-1 68.93±5.30a 24.10±1.29a 5.46±1.13a 50.0 54.2 31.3 30.0 Compared with 0 μmol·L-1, aP<0.05 Compared with 0 μmol•L-1, aP<0.05 higher than in 0 μmol·L-1 Withaferin A (P<0.05). And and Mcl-1 played an important part in the process of25.0 except the early apoptotic rate in 5.0 μmol·L-1 and inducing cellular apoptosis of breast cancer by Withaferin 38.0 31.3 31.3 30.0 33.1 advanced one in 10.0 μmol·L-1 Withaferin A, the apoptotic A, which was proved by the activation of MAPK pathways 23.7 25.0 27.6 rates of A549 cells treated by 5.0, 10.0 and 20.0 μmol·L-1 in cellular experiments. Li et al. (2013) found that 0 0 Withaferin A for 48 h were all markedly higher than those Withaferin A could induce the apoptosis of GBC-SD cells for 24 h, and there were significant differences P( <0.05) in human gallbladder carcinoma by impacting cellular (Table 1). cycles, such as changing the expressions of periodic None

Influence of apoptosis-associated gene expression: elements, etc.. And Roy et al. (2013) discovered that Remission

A549 cells treated by each dosage of Withaferin A (except Withaferin A could influence the cellular proliferation of Radiotherapy 2.5 μmol·L-1) for 48 h were lower in Bcl-2 expression gland so as to inhibit its growth. All studies above and higher in Bax and Cleaved caspase-3 expressions ensured that Withaferin A can inhibit cellular activity of than those in 0 μmol·L-1, respectively, and the differences multiple tumors through various ways. -1 recurrence or Persistence among 5, 10 and 20 μmol·L were significant (P<0.05) In this study, it was found that Withaferin A could chemoradiation Concurrent (Table 2). evidently inhibit A549 cellular proliferation time- and

dosage-dependently in that the inhibiting rate of A549 withtreatment diagnosed Newly Effect of Withaferin A on cellular cycle cellular proliferation increased along with the increase withouttreatment diagnosed Newly The ratios of A549 cells treated by different dosages of dosages and time, which was consistent with the of Withaferin A for 48 h in G0/G1 stage were higher than researches of Withaferin A on other tumors (Hahm et -1 those in 0 μmol·L , while those in S and G2/M stages al., 2013; Roy et al., 2013). Moreover, this study also were obviously lower than in G2/M stage, and there were verified the effect of Withaferin A on A549 cellular significant differences in cellular cycle distribution treated apoptosis, including apoptotic indexes, apoptotic rates and by 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A (P<0.05) apoptosis-associated gene expressions by many methods, (Table 3). which demonstrated that Withaferin A could induce the apoptosis of A549 cells, and had obvious influence on Effect of Withaferin A on PI3K/Akt signal pathway cellular apoptosis in all dosages except 2.5 μmol·L-1, The p-Akt/Akt values of A549 cells treated by 0, and the differences were significant. Bax and cleaved- 2.5, 5.0, 10.0 and 20.0 μmol·L-1 Withaferin A for 48 h Caspase-3 are apoptosis-promoting genes, in which Bcl-2 were (0.32±0.17), (0.26±0.12), (0.18±0.09), (0.12±0.04) is the most common one in inhibiting apoptosis (Liu et and (0.06±0.01), respectively, which were reversely al., 2010; Kim et al., 2012), while the apoptotic condition proportional to dosages, and the differences were is often evaluated by the changes of above 3 protein significant P( <0.05). levels. Additionally, this study suggested that Withaferin A could elevate Bax and cleaved-Caspase-3 levels and Discussion reduce Bcl-2 level, improving the cellular apoptosis on molecular level. Withaferin A is a natural anti-tumor agent, however, PI3K/Akt signal pathways are critical in cellular its mechanism of inhibiting the cellular proliferation of proliferation and apoptosis, which are activated in most multiple tumors is still unclear, which may be correlated tumor cells (Osaki et al., 2004), leading to increased p-Akt, with inducing cellular apoptosis, inhibiting DNA synthesis a formation in phosphorylated Akt induced by PI3K. and impacting cellular signal pathways (Hahm et al., 2013; However, the ratio of p-Akt may decrease if PI3K/Akt Lee et al., 2013). Eun-Ryeong et al (Hahm et al., 2013) signal pathways are not activated. In this study, p-Akt/ proposed that mitogen activited protein kinase (MAPK) Akt decreased after 48 h treatment with Withaferin A, Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 1713 Yong Cai et al indicating that Withaferin A could inhibit the activity of PI3K/Akt signal pathways, therefore, it was predicated that Withaferin A might inhibit the proliferation and apoptosis of A549 cells by suppressing PI3K/Akt signal pathways. In conclusion, Withaferin A can inhibit the proliferation and apoptosis of A549 cells by suppressing PI3K/Akt signal pathways, which has certain prospect in treating NSCLC.

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